Plasma membrane-bound phenolic peroxidase of maize roots: in vitro regulation of activity with NADH and ascorbate

Plant Science ◽  
2003 ◽  
Vol 165 (6) ◽  
pp. 1429-1435 ◽  
Author(s):  
Vesna Hadži-Tašković Šukalović ◽  
Mirjana Vuletić ◽  
Željko Vučinić
Keyword(s):  
Plasma Membrane ◽  
Maize Roots ◽  
Regulation Of Activity ◽  
Membrane Bound ◽  
Roots In Vitro

scholarly journals Morphogenic Effects of Ezrin Require a Phosphorylation-Induced Transition from Oligomers to Monomers at the Plasma Membrane

2000 ◽  
Vol 150 (1) ◽  
pp. 193-204 ◽  
Author(s):  
Alexis Gautreau ◽  
Daniel Louvard ◽  
Monique Arpin
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Wild Type ◽  
Erm Proteins ◽  
Threonine Residue ◽  
Phosphorylation Event ◽  
Membrane Bound

ERM (ezrin, radixin, moesin) proteins act as linkers between the plasma membrane and the actin cytoskeleton. An interaction between their NH2- and COOH-terminal domains occurs intramolecularly in closed monomers and intermolecularly in head-to-tail oligomers. In vitro, phosphorylation of a conserved threonine residue (T567 in ezrin) in the COOH-terminal domain of ERM proteins disrupts this interaction. Here, we have analyzed the role of this phosphorylation event in vivo, by deriving stable clones producing wild-type, T567A, and T567D ezrin from LLC-PK1 epithelial cells. We found that T567A ezrin was poorly associated with the cytoskeleton, but was able to form oligomers. In contrast, T567D ezrin was associated with the cytoskeleton, but its distribution was shifted from oligomers to monomers at the membrane. Moreover, production of T567D ezrin induced the formation of lamellipodia, membrane ruffles, and tufts of microvilli. Both T567A and T567D ezrin affected the development of multicellular epithelial structures. Collectively, these results suggest that phosphorylation of ERM proteins on this conserved threonine regulates the transition from membrane-bound oligomers to active monomers, which induce and are part of actin-rich membrane projections.

scholarly journals Single molecule dynamics of Dishevelled at the plasma membrane and Wnt pathway activation

2019 ◽  
Author(s):  
Wenzhe Ma ◽  
Maorong Chen ◽  
Hong Kang ◽  
Zachary Steinhart ◽  
Stephane Angers ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Wnt Signaling ◽  
Single Molecule ◽  
Wnt Pathway ◽  
Wnt Signaling Pathway ◽  
Membrane Receptors ◽  
Pathway Activation ◽  
Mean Size ◽  
Membrane Bound

AbstractDvl (Dishevelled) is one of several essential non-enzymatic components of the Wnt signaling pathway. In most current models, Dvl forms complexes with Wnt ligand receptors, Fzd and LRP5/6 at the plasma membrane, which then recruits other components of the destruction complex leading to inactivation of β-catenin degradation. Although this model is widespread, direct evidence for this process is lacking. In this study, we tagged mEGFP to C-terminus of dishevlled2 gene using CRISPR/Cas9 induced homologous recombination and observed its dynamics directly at the single molecule level with Total Internal Reflection Fluorescence (TIRF) microscopy. We focused on two questions: 1) What is the native size and the dynamic features of membrane-associated Dvl complexes during Wnt pathway activation? 2) What controls the behavior of these complexes? We found that membrane bound Dvl2 is predominantly monomer in the absent of Wnt (mean size 1.10). Wnt3a stimulation leads to an increase in the total concentration of membrane-bound Dvl2 from 0.08/μm2 to 0.34/μm2. Wnt3a also leads to increased oligomerization which raises the weighted averaged mean size of Dvl2 complexes to 1.4; with 65% of Dvl still as monomers. The driving force for Dvl2 oligomerization is the increased concentration of Dvl2 at the membrane caused by increased affinity of Dvl2 for Fzd, the Dvl2 and Fzd binding is independent of LRP5/6. The oligomerized Dvl2 complexes have greatly increased dwell time, 2~3 minutes compared to less than 1 second for monomeric Dvl2. These properties make Dvl a unique scaffold dynamically changing its state of assembly and stability at the membrane in response to Wnt ligands.Significance StatementCanonical Wnt signaling is one of the most widely distributed pathways in metazoan development. Despite intense genetic and biochemical study for over 35 years, the major features of signaling across the plasma membrane are still poorly understood. Dishevelled serves as an essential bridge between the membrane receptors and downstream signaling components. Attempts to reconstruct the pathway and analyze its biochemical features in vitro have been hampered by Dishevelled’s tendency to aggregate in vitro and to form large aggregates of dubious significance in vivo. To obtain a molecular understanding of the role of Dvl in Wnt signaling, while circumventing these aggregation problems we have expressed a fluorescent tagged Dishevelled in cells at their physiological concentration and quantified the size distribution of Dishevelled before and after Wnt treatment. We found that limited oligomerization in response to the Wnt ligand is very dynamic and provides a key step of signal transduction.

scholarly journals In vitro release of physically separable factors from monocytes that exert opposing effects on erythropoiesis

Blood ◽  
1986 ◽  
Vol 67 (5) ◽  
pp. 1454-1459 ◽  
Author(s):  
L Feldman ◽  
CM Cohen ◽  
N Dainiak
Keyword(s):  
Plasma Membrane ◽  
Membrane Vesicle ◽  
In Vitro Release ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Wide Range ◽  
Dose Dependent Fashion ◽  
Membrane Bound ◽  
Opposing Effects

Abstract In order to investigate the capacity of monocytes to release erythroid burst-promoting activity (BPA), we added media conditioned by homologous monocytes to both serum-free human and serum-restricted murine marrow culture. We found that soluble, membrane vesicle-free culture medium is a potent source of the growth factor. On the other hand, monocyte membranes or exfoliated plasma membrane vesicles elaborate a factor that inhibits erythroid burst formation by up to 100%. Inhibitory activity is expressed in a dose-dependent fashion over a wide range of concentrations (0.001 to 10 micrograms/mL) tested. Experiments with antilymphocyte plasma membrane IgG, which has been shown to neutralize both soluble and membrane-bound lymphocyte-derived BPA in human marrow culture, indicate that the expression of soluble BPA by monocytes is unaffected by these antibodies. Furthermore, while antimembrane IgG is capable of absorbing BPA from LCM supernatants, these antibodies are ineffective in removing BPA from MCM supernatants, suggesting that these two soluble growth factors may be antigenically distinct. Our findings indicate that while monocytes release soluble BPA, they are also a source of membrane-associated factors that exert inhibitory effects on erythropoiesis in vitro.

scholarly journals Human NDR Kinases Are Rapidly Activated by MOB Proteins through Recruitment to the Plasma Membrane and Phosphorylation

2005 ◽  
Vol 25 (18) ◽  
pp. 8259-8272 ◽  
Author(s):  
Alexander Hergovich ◽  
Samuel J. Bichsel ◽  
Brian A. Hemmings
Keyword(s):  
Plasma Membrane ◽  
Regulatory Mechanism ◽  
Membrane Targeting ◽  
Membrane Translocation ◽  
Cancer Types ◽  
Membrane Bound ◽  
Insight Into ◽  
Constitutively Active

ABSTRACT Human nuclear Dbf2-related kinases (NDRs) are up-regulated in certain cancer types, yet their precise function(s) and regulatory mechanism(s) still remain to be defined. Here, we show that active (phosphorylated on Thr444) and inactive human NDRs are both mainly cytoplasmic. Moreover, NDR kinases colocalize at the plasma membrane with human MOBs (hMOBs), which are recently described coactivators of human NDR in vitro. Strikingly, membrane targeting of NDR results in a constitutively active kinase due to phosphorylation on Ser281 and Thr444 that is further activated upon coexpression of hMOBs. Membrane-targeted hMOBs also robustly promoted activation of NDR. We further demonstrate that the in vivo activation of human NDR by membrane-bound hMOBs is dependent on their interaction and occurs solely at the membrane. By using a chimeric molecule of hMOB, which allows inducible membrane translocation, we found that NDR phosphorylation and activation at the membrane occur a few minutes after association of hMOB with membranous structures. We provide insight into a potential in vivo mechanism of NDR activation through rapid recruitment to the plasma membrane mediated by hMOBs.

scholarly journals Phosphorylation of membrane-bound guanylate cyclase of sea urchin spermatozoa.

1986 ◽  
Vol 103 (1) ◽  
pp. 95-101 ◽  
Author(s):  
G E Ward ◽  
G W Moy ◽  
V D Vacquier
Keyword(s):  
Plasma Membrane ◽  
Enzymatic Activity ◽  
Guanylate Cyclase ◽  
Ammonium Hydroxide ◽  
Arbacia Punctulata ◽  
Direct Demonstration ◽  
Seawater Ph ◽  
Sperm Plasma Membrane ◽  
Membrane Bound

When Arbacia punctulata spermatozoa are incubated in seawater containing ammonium hydroxide (pH 8.8), the sperm plasma membrane-bound guanylate cyclase is dephosphorylated, its electrophoretic mobility increases (from an apparent molecular mass of 160 to 150 kD), and its enzymatic activity decreases 3.5-fold. Transfer of these cells into ammonium-free seawater (pH 7.4) results in the rephosphorylation of the cyclase, its reconversion to 160 kD, and recovery of the enzymatic activity lost upon dephosphorylation. This is the first direct demonstration that the activity of membrane-bound guanylate cyclase can be regulated by phosphorylation. A plasma membrane preparation is described that specifically supports the in vitro phosphorylation of the guanylate cyclase. This preparation will be useful in more detailed studies on the relationship between phosphorylation state and enzymatic activity of membrane-bound guanylate cyclase.

scholarly journals The Structural and Functional Implications of Linked SNARE Motifs in SNAP25

2008 ◽  
Vol 19 (9) ◽  
pp. 3944-3955 ◽  
Author(s):  
Li Wang ◽  
Mary A. Bittner ◽  
Daniel Axelrod ◽  
Ronald W. Holz
Keyword(s):  
Plasma Membrane ◽  
Chromaffin Cells ◽  
Close Association ◽  
Living Cells ◽  
Full Length ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Functional Implications ◽  
Membrane Bound

We investigated the functional and structural implications of SNAP25 having two SNARE motifs (SN1 and SN2). A membrane-bound, intramolecular FRET probe was constructed to report on the folding of N-terminal SN1 and C-terminal SN2 in living cells. Membrane-bound constructs containing either or both SNARE motifs were also singly labeled with donor or acceptor fluorophores. Interaction of probes with other SNAREs was monitored by the formation of SDS-resistant complexes and by changes in FRET measured in vitro using spectroscopy and in the plasma membrane of living cells using TIRF microscopy. The probes formed the predicted SDS-resistant SNARE complexes. FRET measurements revealed that syntaxin induced a close association of the N-termini of SN1 and SN2. This association required that the SNARE motifs reside in the same molecule. Unexpectedly, the syntaxin-induced FRET was prevented by VAMP. Both full-length SNAP25 constructs and the combination of its separated, membrane-bound constituent chains supported secretion in permeabilized chromaffin cells that had been allowed to rundown. However, only full-length SNAP25 constructs enabled robust secretion from intact cells or permeabilized cells before rundown. The experiments suggest that the bidentate structure permits specific conformations in complexes with syntaxin and VAMP and facilitates the function of SN1 and SN2 in exocytosis.

Externalization of membrane-bound activities during sheep reticulocyte maturation is temperature and ATP dependent

1987 ◽  
Vol 65 (12) ◽  
pp. 1080-1090 ◽  
Author(s):  
Linda Orr ◽  
M. Adam ◽  
R. M. Johnstone
Keyword(s):  
Plasma Membrane ◽  
Metabolic Process ◽  
Atp Depletion ◽  
Temperature Dependent ◽  
Associated Functions ◽  
Reticulocyte Maturation ◽  
Membrane Bound ◽  
Membrane Components ◽  
Specific Membrane

During the maturation of sheep reticulocytes in vitro, there is release of material that can be pelleted from the cell-free incubation medium by centrifugation at 100 000 × g. This pellet contains activities that are derived from both the plasma membrane and lysosomes. No evidence was obtained for the presence of mitochondrial activities or cytosolic enzyme activities. The release of these activities is ATP and temperature dependent, since reduction of either results in a greater retention of the activities by the cells and a lesser amount in the 100 000 × g pellet. The pelleted material is vesicular in nature, and the production and (or) release of the material are reduced upon ATP depletion or lowering of the temperature. It is concluded that the externalization of specific membrane components is a normal metabolic process that occurs during reticulocyte maturation and represents a means by which reticulocytes shed specific types of membrane-associated functions that are known to decrease during reticulocytes maturation.

scholarly journals In vitro release of physically separable factors from monocytes that exert opposing effects on erythropoiesis

Blood ◽  
1986 ◽  
Vol 67 (5) ◽  
pp. 1454-1459
Author(s):  
L Feldman ◽  
CM Cohen ◽  
N Dainiak
Keyword(s):  
Plasma Membrane ◽  
Membrane Vesicle ◽  
In Vitro Release ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Wide Range ◽  
Dose Dependent Fashion ◽  
Membrane Bound ◽  
Opposing Effects

In order to investigate the capacity of monocytes to release erythroid burst-promoting activity (BPA), we added media conditioned by homologous monocytes to both serum-free human and serum-restricted murine marrow culture. We found that soluble, membrane vesicle-free culture medium is a potent source of the growth factor. On the other hand, monocyte membranes or exfoliated plasma membrane vesicles elaborate a factor that inhibits erythroid burst formation by up to 100%. Inhibitory activity is expressed in a dose-dependent fashion over a wide range of concentrations (0.001 to 10 micrograms/mL) tested. Experiments with antilymphocyte plasma membrane IgG, which has been shown to neutralize both soluble and membrane-bound lymphocyte-derived BPA in human marrow culture, indicate that the expression of soluble BPA by monocytes is unaffected by these antibodies. Furthermore, while antimembrane IgG is capable of absorbing BPA from LCM supernatants, these antibodies are ineffective in removing BPA from MCM supernatants, suggesting that these two soluble growth factors may be antigenically distinct. Our findings indicate that while monocytes release soluble BPA, they are also a source of membrane-associated factors that exert inhibitory effects on erythropoiesis in vitro.

scholarly journals EHBP1 and EHD2 regulate Dll4 caveolin-mediated endocytosis during blood vessel development

2020 ◽  
Author(s):  
Amelia M Webb ◽  
Caitlin R Francis ◽  
Jayson M Webb ◽  
Hayle Kincross ◽  
Keanna M Lundy ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Blood Vessel ◽  
Notch Signaling ◽  
Blood Vessel Development ◽  
Absolute Requirement ◽  
Membrane Bound ◽  
Vessel Development ◽  
Notch Activation

ABSTRACTDespite the absolute requirement of Delta/Notch signaling to activate lateral inhibition during early blood vessel development, many mechanisms remain unclear. Here, we identify EHD2 and EHBP1 as novel regulators of Notch activation in endothelial cells through controlling endocytosis of Delta-like ligand 4 (Dll4). Knockout of EHBP1 and EHD2 in zebrafish produced a significant increase in ectopic sprouts in zebrafish intersomitic vessels during development and a reduction in downstream Notch signaling. In vitro, EHBP1 and EHD2 localized to plasma membrane-bound Dll4 and actin independently of clathrin. Disruption of caveolin endocytosis resulted in EHBP1 and EHD2 failing to organize around Dll4 as well as loss of Dll4 internalization in endothelial cells. Overall, we demonstrate that EHBP1 and EHD2 regulate Dll4 endocytosis by anchoring caveolar endocytic pits to the actin cytoskeleton.

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